The myelin sheath covers the axon in segments that are separated by the nodes of Ranvier. In the underlying axons Na+ channels are clustered at the nodes of Ranvier, separated by a specialized axoglial paranodal junction (PNJ) from K+ channels that are found at the nearby juxtaparanodal region (JXP). This organization is essential for the saltatory movement of the nerve impulses, and its disturbance results in pathophysiological changes often seen in demyelinating human disorders. In the peripheral nervous system (PNS), myelinating Schwann cells regulate this precise organization of the axonal membrane through unknown mechanisms. We have previously identified several cell adhesion molecules (CAMs), including gliomedin, Caspr, Caspr2, and Necl4, which mediate axoglial contact at specific sites along the longitudinal axis of the myelin unit. These CAMs play important roles in the organization of myelinated axons: gliomedin cluster Na+ channels at nodes, Caspr is involved in the generation of a membrane barrier at the PNJ that restricts the distribution of Na+ and K+ channels to the nodes and JXP, respectively, Caspr2 serves as a scaffold that maintains K+ channels at the JXP, and Necl proteins organize the internodal membrane. The objective of the proposed research is to study the molecular mechanisms underlying the function of these axoglial CAMs in the generation of functional domains along myelinated axons. First, complementation experiments will be carried out using myelinating Schwann cells/neuron cultures isolated from gldn-/- mice to determine how the channels clustering activity of gliomedin is being regulated. Secondly, a transgenic rescue approach will be taken to examine the hypothesis that the PNJ restricts the distribution of Na+ channels to the nodal gap by forming a membrane barrier, which depends on the presence of the axonal cytoskeleton. Third, a similar in vivo approach will be taken in order to examine the mode of interaction between Caspr2 and TAG-1 and to reveal the underlying mechanisms by which these molecules recruit and retain Kv1 channels at the JXP. Finally, we will study the role of Necl proteins in the organization of myelinated axons by analyzing the morphology and the molecular organization of peripheral nerves of mutant mice lacking Necl4, as well as mice lacking the cytoskeletal linker protein 4.1G, which colocalizes and associates with Necl4 in myelinating Schwann cells. Altogether, the proposed experiments will provide important information about the coordinated differentiation of axons and myelin-forming cells, which allow myelinated fibers to maximize their conduction velocity. Better understanding these mechanisms may thus lead to new routes to the restoration of function in demyelinated axons and may prove useful in the design of therapeutical strategies for demyelinating disorders. PUBLIC HEALTH RELEVANCE: A functional nervous system requires the precise localization of ion channels at distinct membrane domains, where they are optimally positioned for their function. How such domains are being generated and maintained is of particular importance for our understanding of the pathological mechanisms operating in demyelinated diseases, in which reorganization of the axonal membrane and the redistribution of ion channels affects the physiological properties of the nerve fibers. Uncovering the mechanisms by which myelinating glial cells control the clustering of ion channels along myelinated axons is highly significant given the prevalence of neurodegenerating diseases that result from demyelination or alteration in myelin, such as multiple sclerosis and a number of inherited neuropathies. Our studies on the generation of functional myelinated nerves may lead to new routes to the restoration of function of demyelinated nerves.